Hybrid (intravenous and oral) administration of vinorelbine plus cisplatinum followed by oral vinorelbine as first-line therapy of advanced non-small cell lung cancer: a phase II study.

BACKGROUND: The combination of alternate i.v./oral (hybrid) administration of vinorelbine (VNR) plus cisplatin (CDDP), followed by oral VNR, could result in a more suitable first-line regimen for patients (pts) with advanced non-small cell lung cancer (aNSCLC) in the outpatient setting. METHODS: The induction treatment consisted of CDDP 80 mg/m(2) i.v. and VNR 25 mg/m(2) i.v. day 1 and VNR 60 mg/m(2) oral day 8, every 3 weeks for 4 courses. A dose escalation of VNR to 80 mg/m(2) oral from day 8 of the second course and to 30 mg/m(2) i.v. from day 1 of the third course was planned in the absence of G3-4 toxicity. Pts with disease control after 4 courses underwent consolidation treatment with oral VNR 80 mg/m(2) days 1 and 8 every 3 weeks up to intolerance or progression. RESULTS: Fifty-three pts entered the study: 80% males; median age 63 years (range 43-71); median ECOG PS 0 (range 0-1); histotype: adenocarcinoma 59%, epidermoid 31%, undifferentiated 10%; disease stage: IIIB 22%, IV 70%, recurrent disease 8%. The objective response was as follows: 1 (2%) CR, 20 (38%) PR, 16 (30%) SD, 11 (21%) PD and 5 (9%) pts were not assessable. Median TTP and OS were 6 and 10 months, respectively. G3-4 neutropenia was observed in 23 and 24% of pts in the induction and in the consolidation phases, respectively, with febrile neutropenia in 6 pts (11%) and 2 (8%), respectively. G3-4 non-haematological toxicity was rare, being represented by nausea-vomiting and neurotoxicity in 3 pts (6%) in the induction phase. CONCLUSIONS: This combination regimen including hybrid administration of VNR plus CDDP is feasible, tolerable and effective as a first-line treatment in pts with aNSCLC.

Cardiac toxicity of trastuzumab in metastatic breast cancer patients previously treated with high-dose chemotherapy: a retrospective study.

HER-2 overexpression is associated to a poor prognosis in high-risk and metastatic breast cancer (MBC) patients treated with high-dose chemotherapy (HDC). HER-2 status is also a predictive factor and when trastuzumab is administered in combination with or sequentially to chemotherapy, a significant disease-free and/or overall survival improvement has been observed in HER-2+ early and MBC. Unfortunately, in both settings, trastuzumab is associated with an increased risk of cardiac dysfunction (CD). We have reviewed the clinical charts of HER-2-overexpressing MBC patients treated with trastuzumab after HDC. Age, baseline left ventricular ejection fraction (LVEF), radiation therapy on cardiac area, exposure to anthracycline, single or multiple transplant, high-dose agents, trastuzumab treatment duration were recorded as potential risk factors. In total, 53 patients have been included in the analysis. Median LVEF at baseline was 60.5%; at the end of trastuzumab (data available for 28 patients only), it was 55% (P = 0.01). Five out of the 28 (17.9%) patients experienced CD. Two out of 53 (3.8%) patients developed a congestive heart failure. Age > or = 50 years and multiple transplant procedure were potential risk factors for CD. The overall incidence of CD observed in this population of HER-2+ MBC patients treated with trastuzumab after HDC is not superior to that reported with concomitant trastuzumab and anthracyclines. However, patients with age > or = 50 years or receiving multiple course of HDC should be considered at risk for CD.

Incidence of human cytomegalovirus infection in patients with refractory solid tumors receiving nonmyeloablative allogeneic stem cell transplants versus recipients of standard SCT for hematologic malignancies.

Human cytomegalovirus (HCMV) infection is the most frequent infectious complication after conventional allogeneic stem cell transplantation (alloSCT). From December 1998 to December 2002, we prospectively monitored HCMV reactivation in 59 patients affected by solid tumors and undergoing nonmyeloablative alloSCT (NST). Patients were allografted from HLA-identical sibling donors after fludarabine/cyclophosphamide-based conditioning regimens. Seventeen (28.8%) of 59 patients presented with HCMV antigenemia, and 14 received ganciclovir, with successful HCMV clearance in all cases. No patient developed HCMV viremia or disease. The median time to HCMV reactivation was 54 days (range, 16-245 days) after NST. These patients were compared with a cohort of hematologic patients who were treated with conventional myeloablative alloSCT. Matching criteria included HCMV risk group, stem cell source, donor type, and age. In the myeloablative group, HCMV active infection was observed in 47 (85.4%) of 55 patients at a median time of 30 days (range, 13-64 days) after alloSCT, and HCMV infection occurred more frequently ( P < .001) and earlier ( P = .001) than in NST patients. Patients affected with solid tumors undergoing NST had a reduced and delayed incidence of HCMV active infection.

Randomized, controlled, multicenter phase III trial of standard-dose fluorouracil-epirubicin-cyclophosphamide (FEC), compared with time-intensive FEC (FEC-G) and mitoxantrone-methotrexate-mitomycin C (MMM-G) in metastatic breast carcinoma.

The purpose of this multicenter phase III trial was to assess the impact of a time-intensification of FEC (fluorouracil, epirubicin, cyclophosphamide) and MMM (mitoxantrone, methotrexate, mitomycin C) regimens, supported by lenograstim (G-CSF) on the objective response rate, time to progression and survival of patients with chemotherapy-naive metastatic breast cancer (mbc). Women with mbc were randomized to receive as first-line chemotherapy either standard-dose FEC (all doses in mg/m2): arm A (500, 75, 500 every 21 days), or time-intensified FEC-G: arm B (500, 75, 500 every 14 days), or time-intensified MMM-G: arm C (mitoxantrone 10, methotrexate 35 every 14 days and mitomycin C 10 every 28 days), both with support of lenograstim (G-CSF 150 microg/m2/day s.c. for 10 days). All study treatments were administered for six cycles. Eligible female patients were in the 31-70 year range with histologically proven mbc, and measurable or evaluable disease. An intent-to-treat analysis was performed. The overall response rate (CR + PR, intent-to-treat analysis) was significantly improved in the time-intensified FEC-G regimen (69%) in comparison with standard-dose FEC (41%), p=0.002. Time-intensified MMM-G (51%) did not lead to a significant improvement in the response rate. The percentage of complete responses was significantly higher in the FEC-G arm as compared to standard-dose FEC (17% vs. 4.7%; p=0.002). The median duration was longer in the intensified-dose arms without, however, achieving a statistically significant improvement. The median time to progression (TTP), and the median survival time did not differ between the three treatment arms. Grade 3-4 leukopenia was significantly higher (p<0.001) in the standard FEC regimen-treated patients. Thrombocytopenia was significantly higher (p<0.001) in both intensified regimens. Alopecia and mucositis were significantly more frequent in both anthracycline-containing regimens (p=0.003). Other hematological and non hematological toxicities were similar in the 3 treatment arms. The increase of dose-intensity of both FEC and MMM regimens improved activity, but not efficacy as compared to standard FEC regimen in our group of chemotherapy-naive, metastatic breast cancer patients.

Low incidence of secondary myelodysplasia and acute myeloid leukemia after high-dose chemotherapy as adjuvant therapy for breast cancer patients: a study by the Solid Tumors Working Party of the European Group for Blood and Marrow Transplantation.

BACKGROUND: To determine the incidence of secondary myelodysplasia (sMDS) or acute myeloid leukemia (AML) in node-positive breast cancer patients who received high-dose chemotherapy (HDCT) followed by autologous stem-cell support as adjuvant therapy. PATIENTS AND METHODS: The incidence of sMDS/AML was retrospectively assessed in 364 node-positive breast cancer patients who received HDCT followed by autologous stem-cell support as adjuvant therapy between November 1989 and December 1997 and were reported to the European Group for Blood and Marrow Transplantation registry. RESULTS: The median age of the patients was 45 years (range 22-62 years). Two hundred and ninety-one patients received peripheral blood stem cells and 55 patients received autologous bone marrow as stem-cell support. The most frequently used conditioning regimen was the STAMP-V regimen (32%), followed by melphalan-thiotepa (22%) and melphalan-mitoxantrone-cyclophosphamide (21%). The 5-year probability of overall survival is 71% (95% CI 65% to 77%). After a median follow-up of 48 months (range 1-108 months) only one case of AML was observed, resulting in a crude incidence of 0.27%. This case of AML was observed 18 months after HDCT consisting of three cycles of epirubicin and cyclophosphamide with a cumulative dose of epirubicin 960 mg and cyclophosphamide 19 g. The French-American-British type of AML was M4, and the cytogenetic analysis showed a translocation t(9;11)(p22;q23). After complete remission following high-dose cytarabine and idarubicin the patient relapsed and died. CONCLUSIONS: In contrast to patients with malignant lymphoma there seems to be no increased risk of sMDS/AML after HDCT in breast cancer. Continued monitoring is required to confirm this low incidence after a longer follow-up period.

Cytotoxic chemotherapy preceding apheresis of peripheral blood progenitor cells can affect the early reconstitution phase of naive T cells after autologous transplantation.

Transient T cell immunodeficiency is a common complication following hematopoietic stem cell transplantation. In breast cancer patients transplanted with autologous peripheral blood progenitor cells (PBPC) harvested after cytotoxic treatment with either cyclophosphamide or epirubicin plus paclitaxel, we evaluated T cells infused in grafts and in peripheral blood during the early reconstitution phase. We found that PBPC grafts harvested after treatment with epirubicin plus paclitaxel contained substantially larger numbers of T cells with less altered composition than after cyclophosphamide. Three months after high-dose cytotoxic chemotherapy, the numbers and the kinetics of circulating naive T cells, but not of memory and CD28- T cells, correlated positively with the number of naive T cells infused PBPC grafts. Finally, retrospective analysis of two cohorts of patients transplanted in different clinical settings with PBPC grafts harvested following cyclophosphamide or epirubicin plus paclitaxel showed apparently different susceptibilities to develop endogenous varicella zoster virus reactivation in the first year after high-dose cytotoxic chemotherapy. On the whole, these data indicate that number and composition of T cells in PBPC grafts vary according to the former cytotoxic therapy, and suggest that autologous transfer of T cells may accelerate the early T cell reconstitution phase and possibly ameliorate immune competence in patients rendered lymphopenic by high-dose chemotherapy.

Diverse expansion potential and heterogeneous avidity in tumor-associated antigen-specific T lymphocytes from primary melanoma patients.

While tumor-associated antigen (TAA)-specific CD8(+) T lymphocytes have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We looked for circulating cytotoxic T lymphocytes (CTL) directed against Melan-A / MART1, tyrosinase, gp100 and MAGE-3 antigens in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA-A2 tetramers. In five out of six cases high numbers of CD8(+)/tetramer(+) cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan-A/MART1 and tyrosinase) were contemporaneously present. The TAA-specific cells could represent as much as 1/220 T lymphocytes in the circulating CD8(+) population. When tetramers were used to monitor the in vitro expansion of TAA-specific CTL precursors upon antigen-specific stimulation, a diverse expansion potential was evidenced in CTL from the different donors and, more strikingly, in CTL specific for the different TAA. Melan-A/MART1-specific CTL clones derived from two patients exhibited a broad range of avidity. Only the highest avidity clones, representing about 50 % of the cases analyzed, were tumor specific. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall clone avidity.

Four-day infusion of fluorouracil plus vinorelbine as salvage treatment of heavily pretreated metastatic breast cancer.

AIMS: Anthracyclines-taxanes containing regimens are widely used for breast cancer treatment both in neoadjuvant-adjuvant setting and in metastatic disease. Recently high-dose chemotherapy (HDC) with autologous stem cell support has been introduced as adjuvant treatment for high-risk primary breast cancer and for selected subsets of women with metastatic disease. Therefore, salvage treatment for previously treated patients with progressive disease becomes even more problematic. A regimen of continuous infusion of fluorouracil (FU) and vinorelbine (VNR) has been evaluated in heavily pretreated metastatic breast cancer patients. PATIENTS AND METHODS: Forty-eight women, median age 52 years, with previously treated breast cancer entered the study. All but one received more than one line of prior systemic chemotherapy for metastatic disease. Furthermore 14 women had undergone HDC with peripheral blood progenitor cells transplantation in adjuvant setting (6 pts), or metastatic disease (8 pts). Treatment consisted of four-day infusion of FU (1000 mg/m2/day) plus VNR (20 mg/m2/i.v. day 1 and 5), recycled every 3 weeks for a total of six courses. Drugs administration was discontinued for G4 toxicity, tumor progression or patient's refusal. RESULTS: Twenty PR and four CR for an overall response rate of 50% (95%C.I. 36-64%) were recorded. The therapeutic efficacy of the tested regimen was documented both in patients unresponsive to previous anthracyclines-taxanes combinations and in those relapsing after HDC. The median duration of response was 9 months and median survival 16 months. One third of patients experienced Grade-3 stomatitis-mucositis, hematological toxicity was mild and no cardiac toxicity was observed. Twenty-five women (52%) suffered from infusion-related phlebitis (in half of patients a central venous device was necessary at some point of the treatment program). CONCLUSIONS: The combination of FU infusion and VNR i.v. is an effective salvage treatment for heavily pretreated metastatic breast cancer patients, and may represent a valid alternative when other cytotoxic regimens are not feasible.

Negative immunomagnetic purging of peripheral blood stem cell harvests from breast carcinoma patients reduces tumor cell contamination while not affecting hematopoietic recovery.

BACKGROUND: Because tumor contamination of hematopoietic stem cell grafts may influence the outcome in breast carcinoma (BC) patients undergoing high dose chemotherapy (HDC), several ex vivo procedures for the purging of autologous harvests have been investigated. The authors studied the presence of epithelial tumor cells and the growth of hematopoietic progenitors in peripheral blood stem cell (PBSC) collections from patients with metastatic breast carcinoma before and after a purging procedure performed by a negative immunomagnetic BC cell separation. METHODS: Eighteen patients entered the study. Tumor contamination was assessed by conventional immunocytochemistry (ICC) and by a liquid culture assay developed in the study laboratory. Committed and more primitive hematopoietic progenitors were quantitated before and after the negative selection. Ten patients received HDC with purged PBSC support. RESULTS: Before purging, 4 of 18 PBSC collections were found to be contaminated by liquid culture; among these samples, only 1 was positive by ICC. Three of the four positive collections, including the ICC positive sample, became negative after immunomagnetic selection whereas BC cells still were present after the procedure in one harvest. A high recovery of both primitive and mature hematopoietic progenitors was found after the purging procedure. Patients receiving purged PBSC after myeloablation had a prompt and complete hematopoietic reconstitution, and no graft failure was observed at a median follow-up of 1 year. CONCLUSIONS: The preliminary results of the current study suggest that negative selection of BC cells is able to purge PBSC effectively while having no apparent affect on hematopoietic progenitor recovery in vitro and in vivo.

Transfusion of platelet concentrates cryopreserved with ThromboSol plus low-dose dimethylsulphoxide in patients with severe thrombocytopenia: a pilot study.

We have recently reported the possibility of supporting the phase of severe thrombocytopenia after high-dose chemotherapy (HDC) and stem cell transplantation using 5% dimethylsulphoxide (DMSO)-cryopreserved autologous platelet concentrates (PCs). The aim of the present study was to evaluate the therapeutic potential of ThromboSol (a recently developed platelet storage solution) plus PCs cryopreserved in 2% DMSO in patients undergoing myeloablative chemotherapy and autologous transplantation. PCs were collected from 14 women with breast cancer by a single plateletapheresis and cryopreserved in ThromboSol/2% DMSO by either direct insertion in a -80 degrees C freezer or in liquid nitrogen after computer-controlled rate (CR) freezing. When required, PCs were thawed, centrifuged to remove the cryoprotectants and transfused. In vitro studies on thawed platelets showed loss of epitopes of surface glycoproteins and a marked reduction of functional activity compared with fresh platelets. Transfusion of CR-frozen PCs was associated with a mean 1 h corrected count increment (CCI) of 9.2 +/- 5.4 x 109/l and only one allogeneic PC was required in this group. In contrast, six out of seven patients required additional allogeneic transfusions in the -80 degrees C group (CCI = 2.7 +/- 1.4 x 109/l). ThromboSol-treated PCs have the ability to overcome thrombocytopenia if processed by a CR freezing protocol, but appear ineffective when frozen by direct placing at -80 degrees C.

Pulmonary function and complications following chemotherapy and stem cell support in breast cancer.

Pulmonary complications are frequent in patients treated with high-dose chemotherapy and autologous bone marrow transplantation for breast cancer or other solid tumours. This study analyses the development of lung toxicity, changes in respiratory function and occurrence of clinical symptoms in a group of 24 patients (mean age 46+/-7 yrs) who underwent high-dose sequential chemotherapy (HDS) with autologous peripheral blood stem cell (PBSC) support for high risk breast cancer. Clinical examination, chest radiography and lung function tests were performed before the HDS and 1 and 3 months following transplantation. Only one patient developed acute interstitial pulmonary disease which resolved after prednisone therapy. No patients developed infectious complications after transplantation. Baseline respiratory function was normal for most of the parameters. Only lung diffusing capacity of the lung for carbon monoxide (TL,CO) and maximal inspiratory pressure were below the normal range. Following PBSC transplantation only one patient had an altered vital capacity while 72.3% of patients had reduced TL,CO values at 1 month and 54.5% at 3 months after transplantation. Maximal expiratory flow at 25% forced vital capacity, TL,CO and maximal expiratory pres-sure were significantly reduced after 1 month but recovered slightly by 3 months. Arterial oxygen tension between baseline and both follow-up evaluations declined significantly in patients seropositive for human cytomegalovirus. It is concluded that this high-dose sequential chemotherapy regimen is acceptably safe since no pulmonary related mortality or respiratory infectious complications were observed. The only lung function alteration induced was an isolated diffusing capacity of the lung for carbon monoxide impairment, clinically negligible and partially recovered within 3 months.

Minimal tumor contamination of hematopoietic harvests from breast cancer patients can be easily detected by liquid culture assay.

BACKGROUND: Recurrence after PBSC transplantation in breast cancer (BC) patients may be related to the reinfusion of tumor cells contaminating the graft. We have developed a liquid culture (LC) method for the identification of viable epithelial tumor cells in PBSC collections. METHODS: Mononuclear fraction from PBSC harvests of BC patients undergoing high dose chemotherapy (HDC) (adjuvant setting n = 60, metastatic disease n = 30) were seeded in petri dishes containing round cover slips. Cells were cultured for 3 weeks, then cover slips were stained with the pan-cytokeratin A45-B/B3 mAb and scored under a light microscope. Samples were considered positive when more than one adherent cell or a cluster of cells staining bright red was present. Results were compared with those obtained on cytospins prepared directly from the PBSC harvest. Specificity of the method was tested on lymphoma patients, collections: all were negative. The sensitivity, evaluated by serial dilutions of CG5 BC cell line, was 1 epithelial cell in 10(6) mononuclear cells. RESULTS: The percentage of positivity was superimposable in the two groups (adjuvant 25%, metastatic 24%). However, a significantly higher proportion of positive samples from metastatic vs adjuvant patients has shown the presence of tumor clusters (86% vs 33%, p = 0.063). In 21% of all samples a discrepancy with the results obtained by immunocytochemical analysis (ICC) was found, mostly due to liquid-culture-positive/ICC-negative PBSCs. DISCUSSION: Our data suggest that LC assay may enhance the identification of viable disseminated epithelial tumor cells in PBSC grafts and might provide insights about their growth capacity.

Efficacy of epirubicin/paclitaxel combination in mobilizing large amounts of hematopoietic progenitor cells in patients with metastatic breast cancer showing optimal response to the same chemotherapy regimen.

BACKGROUND AND OBJECTIVE: Based on our preliminary experience, we have further evaluated the capacity of the paclitaxel/epirubicin combination (at the dose of 175 and 90 mg/m(2), respectively) plus G-CSF to mobilize hematopoietic progenitors into the circulation. DESIGN AND METHODS: The study was conducted in a homogeneous cohort of 25 stage IV breast cancer patients showing response to three cycles of the same chemotherapy regimen and who were included in a high-dose chemotherapy program. RESULTS: In most cases (68%) more than 5_10(6) CD34+ cells/kg b.w. (the threshold fixed in our study) were collected by a single leukapheresis, 28% and 4% of patients requiring 2 and 3 procedures, respectively. Based on the CD34+ cell count in the peripheral blood, most of the leukaphereses (53%) were performed on day 11 after chemotherapy. More than 50 CD34+ cells/mL along with a preleukapheresis WBC count between 10 and 20_10(9)/L predicted that only a single harvest would be required in 100% of cases. The evaluation of the clonogenic potential of collected cells showed that a large number of committed colony-forming cells (CFCs) and more primitive long-term culture-initiating cell (LTC-IC) hematopoietic progenitors were present in 20 harvests studied. INTERPRETATION AND CONCLUSIONS: These data demonstrate that the epirubicin/paclitaxel combination followed by G-CSF, besides being a very active regimen in MBC, is effective in releasing large amounts of progenitor cells into the circulation which can then be safely employed to support myeloablative regimens.

Poor outcome of patients with resectable breast cancer receiving adjuvant high-dose sequential chemotherapy following preoperative treatment.

BACKGROUND/OBJECTIVES: The prognosis of resectable high risk breast cancer (BC) patients (N+ > 10) is poor with a five-year disease-free survival (DFS) after standard adjuvant ADM/CMF chemotherapy (CT) of about 40%. An improvement in survival has been reported when high-dose chemotherapy with autologous stem cell support is given. It has been recently suggested that nodal status and the degree of pathological remission following preoperative CT administered in patients harbouring tumors larger than 3 cm represent the most important prognostic factors for DFS. Since no data are available regarding the impact of primary CT in the high dose CT adjuvant setting, we retrospectively evaluated the efficacy of administering megadoses of cytotoxic drugs with stem cell support in the subgroup of patients showing poor response to preoperative CT., PATIENTS AND METHODS: Fourteen women with high risk BC, N+ > 10 and tumor size > 3 cm following antracyclin-based primary CT, received high dose sequential chemotherapy (HDS). The median number of positive axillary nodes at surgery was 18 and tumor size was greater than 5 cm in 6 patients. HDS chemotherapy consisted of cyclophosphamide (7 gr/m2), methotrexate (8 gr/m2) plus vincristin (2 mg), 2 courses of carboplatin (360 mg/m2), and Thiotepa (600 mg/m2) plus L-PAM (160 mg/m2) as final myeloablative regimen requiring stem cell support. RESULTS: At a minimum follow up of 12 months (median 18 months, range 12-40) 5 patients remained disease free (36%) and 9 (64%) have relapsed (7 within the first 10 months). CONCLUSION: Our retrospective analysis suggests that BC patients showing poor response to primary CT might fail to achieve the benefits expected from high dose intensification.

Relationship between size and thiazole orange fluorescence of platelets in patients undergoing high-dose chemotherapy.

The reticulated platelet count relies upon the assumption that newly formed platelets contain a residual amount of RNA which selectively binds the dye thiazole orange (TO) and greatly enhances its fluorescence signal. It has, however, recently been shown that almost half of the platelet TO-signal is derived from the labelling of dense-granule nucleotides. It is therefore possible that the higher TO fluorescence of young platelets partially derives from the higher granule content due to their larger volume. To investigate the relationship between platelet size and TO fluorescence we studied 13 patients with high-risk breast cancer undergoing high-dose chemotherapy. Mean platelet volume, platelet distribution width, platelet-large cell ratio, membrane content of glycoprotein Ib and IIb-IIIa and platelet aggregation were significantly greater during resolution than during development of thrombocytopenia, suggesting a prevalence of young and old platelets respectively. Mean TO fluorescence per cell was higher in the platelet population enriched in young cells than in that enriched in old cells, but this difference was no longer observed when the ratio TO signal/platelet size was examined. Moreover, RNase treatment and platelet degranulation reduced TO fluorescence to a similar extent in platelet populations enriched in young or old cells. Therefore our data suggest that the higher TO signal of young platelets is derived, to a significant extent, from their larger volume and granule content.

Autologous platelet collection and storage to support thrombocytopenia in patients undergoing high-dose chemotherapy and circulating progenitor cell transplantation for high-risk breast cancer.

OBJECTIVES: The use of circulating progenitor cell support following high-dose chemotherapy for malignancies decreases but does not entirely abolish platelet transfusion requirement. We investigated the feasibility of supporting the posttransplant thrombocytopenic phase exclusively with autologous platelets collected by apheresis and cryopreserved. METHODS: 25 patients underwent plateletpheresis during the platelet rebound occurring after high-dose cyclophosphamide. Autologous platelets were cryopreserved in 5% dimethylsulfoxide, thawed and transfused during the aplastic phase after the myeloablative regimen whenever clinically required. RESULTS: A single plateletpheresis was carried out in all patients, allowing the harvest of a platelet concentrate with a mean value of 7.7 x 10(11) platelets. No significant procedure- or transfusion-related side effects were recorded. Mean platelet recovery after freezing and thawing was 63% and the mean number of platelet reinfused was 4.8 x 10(11); 23 of 25 patients were fully supported with autologous platelets. CONCLUSION: Plateletpheresis performed in our selected group of patients was found to be a safe and effective procedure to collect large amounts of autologous platelets; the numbers obtained proved to be sufficient for the transfusion demand of almost all patients.

Epithelial tumour cell detection and the unsolved problems of nested RT-PCR: a new sensitive one step method without false positive results.

Sensitive detection of circulating epithelial cancer cells might have important therapeutic and prognostic implications in patients with breast cancer (BC) receiving high-dose chemotherapy and PBSC support. We have compared the specificity and sensitivity of the recently developed 'one tube' reverse transcriptase PCR (RT-PCR) assay with the more widely used nested RT-PCR method for detection of cytokeratin 19 (CK19)-positive cells. The analysis of 30 control samples provides evidence that one tube RT-PCR is highly specific in contrast to the nested method which showed 23% false positive results. The sensitivity of both techniques to detect tumour contamination was 10(-6). PBSC harvests from 45 BC patients were tested with both RT-PCR methods and the results were compared with immunocytochemistry (ICC). The five samples found positive by ICC were also positive by one tube RT-PCR; in addition, 11 more samples were positive by one tube RT-PCR analysis. The greater number of PBSC found positive by one tube RT-PCR might be due to the larger number of cells analysed. We conclude that one tube RT-PCR is sensitive and reveals no false positive results. This method is less time consuming than the nested one, technically simpler and should be considered for tumour cell detection.

Autologous platelet transfusion in patients receiving high-dose chemotherapy and circulating progenitor cell transplantation for stage II/III breast cancer.

BACKGROUND AND OBJECTIVE: Concerns about the risk of transfusion therapy are driving towards new strategies which are designed to minimize exposure to allogeneic blood products. We aimed to find out whether it is possible to support the phase of thrombocytopenia following high-dose chemotherapy (HDC) and circulating progenitor cells (CPC) transplantation by autologous platelet concentrates (PC). DESIGN AND METHODS: PC were collected from 32 patients undergoing HDC and CPC transplantation for stage II/III breast cancer. A single plateletpheresis was performed at rebound after high-dose cyclophosphamide, when platelet count exceeded 250 x 10(9)/L. PC were cryopreserved in 5% DMSO after controlled-rate freezing and stored in liquid nitrogen. In vitro studies of cryopreserved platelets (aggregation, ATP release and change of mean platelet volume induced by EDTA) were performed. When platelet counts dropped below 20 x 10(9)/L following HDC (thiotepa 600 mg/m2, L-PAM 160 mg/m2) and CPC transplant (CD34+ cells > 5 x 10(6)/kg), PC were thawed in a 37 degrees C water bath, centrifuged to remove DMSO, resuspended in autologous plasma and reinfused within one hour. RESULTS: Large quantities of platelets were harvested in all patients (median 6.6 x 10(11), range 4.8-12.2). In vitro studies showed preserved platelet function as compared to both fresh platelets and standard PC. Twenty-eight out of 32 patients received autologous PC. At the time of transfusion most of the patients were febrile (> 38 degrees C) and had mucositis > G2. The median number of platelets reinfused was 3.8 x 10(11) (range 2.0-8.1) with a median loss during the freeze-thaw-wash procedure of 37%. Autotransfusion was able to maintain platelet count above 20 x 10(9)/L in most patients, with a corrected count increment > 7.5 in 20 cases. Four patients required one additional allogeneic transfusion, two because of a poor increment and two due to a late-occurring epistaxis. No side effects related to PC infusion were recorded. Sixteen control patients who received the same HDC and a similar number of CD34+ cells required a total of 17 allogeneic PC units (1 patient did not require platelet transfusion). INTERPRETATION AND CONCLUSIONS: Our data demonstrate that large doses of autologous platelets can easily be collected and safely administered to support the period of thrombocytopenia in patients undergoing HDC and CPC transplantation. Autologous PC in these patients can abrogate the risks deriving from allogeneic platelet transfusion.